[0001] The present invention relates to methods for increasing the resistance of a plant
to a plant RNA virus and means for obtaining RNA virus-resistant plants.
[0002] The impact of plant viruses on the reduction of crop yields is major. Its cost is
estimated at 60 billion U.S. dollars annually in the world (FAO, 2012). One of the
most important characteristics of plant viruses is that 80% of them are RNA viruses
(Mandahar, 2006). RNA viruses infect plants of economic importance, such as wheat,
corn, cabbages, tobacco, potatoes, peanuts or the cocoa tree. There is therefore a
need for innovative strategies against RNA viruses that are responsible for significant
declines in crop yields.
[0003] Plant RNA viruses are characterized by the frequent presence of a tRNA-like structure
(TLS) at the 3' end of their genomic RNA. A tRNA-like structure is an RNA sequence
mimicking a tRNA and capable of being aminoacylated by an amino acid and therefore
of covalently bonding said amino acid residue at the 3' position. The aminoacylation
of TLS demonstrates that their structures are sufficiently similar to those of tRNAs
to be recognized by tRNA binding enzymes (see for review Dreher, 2009 and 2010). The
presence of TLS and/or their aminoacylation are crucial for these viruses. Aminoacylatable
3' TLS may be involved in virus replication in the translation of their RNA and their
packaging (Dreher, 2009). Work performed
in vitro showed long ago that plant virus holding aminoacylatable 3' TLS could be recognized
and cleaved by RNase P enzymes (Guerrier-Takada
et al., 1988).
[0004] In plants, tRNAs are synthesized as of immature precursors. One of the key steps
of their maturation involves the cleavage of an additional 5' nucleotide sequence.
This cleavage is performed by an endonuclease called RNase P. Until very recently,
all characterized RNase P were ribonucleoproteins (RNP), containing an RNA holding
catalytic activity (Altman, 2007). However, a novel type of RNase P has recently been
discovered in eukaryotes. This enzyme, called PRORP (protein-only RNase P), is of
peptide nature and does not require RNA for its function (Gobert
et al., 2010). PRORP enzymes are particularly important in plants because unlike animals
or yeast, they have completely replaced RNPs for RNase P activity (Gutmann
et al., 2012). PRORP enzymes are characterized by the presence of an RNA binding PPR domain,
a metallonuclease domain holding the actual catalytic activity of the protein, as
well as addressing sequences to the organelles (MTS) or the nucleus (NLS). In the
plant model
Arabidopsis thaliana, three PRORP enzymes are found. PRORP 1 is localized in mitochondria and chloroplasts
while PRORP2 and PRORP3 are located in the nucleus. Moreover, beyond tRNAs, it was
shown that PRORP enzymes are capable both
in vitro (Gobert
et al., 2010) and
in vivo (Gutmann
et al., 2012) to cleave 3' tRNA-like structures (TLS).
[0005] The inventors have constructed a mutant of
A. thaliana PRORP2 protein (At2g16650) (called CytoRP). CytoRP is the result of the deletion
of the first 24 amino acids (corresponding to the nuclear localization signal domain)
of PRORP2. The inventors have constructed genetically transformed
A. thaliana plants expressing CytoRP. The selected plants expressed CytoRP which is located in
the cytosol and holds RNase P activity. This activity leads to the cleavage of the
aminoacylatable 3' tRNA-like structure (TLS) of plant RNA viruses and thus generates
plants with increased resistance to RNA viruses.
[0006] Accordingly, the present disclosure discloses a method for increasing the resistance
of a plant to a plant RNA virus, wherein said method comprises expressing in said
plant a mutant protein-only RNase P enzyme (hereinafter called CytoRP), and wherein
said CytoRP:
- is a protein-only RNase P enzyme comprising neither a nuclear localization signal
(NLS) domain nor an organelle targeting sequence (MTS) domain, and
- is able to cleave the aminoacylatable 3' tRNA-like structure (TLS) of a plant RNA
virus.
[0007] The present invention concerns a method for increasing the resistance of a plant
to a plant RNA virus, said plant RNA virus comprising an aminoacylatable 3' tRNA-like
structure, wherein said method comprises expressing in said plant a mutant protein-only
RNase P (PRORP) enzyme (hereinafter called CytoRP), and wherein said CytoRP:
- is a PRORP enzyme comprising neither a nuclear localization signal (NLS) domain nor
an organelle targeting sequence (MTS) domain and comprising the sequences SEQ ID NO:
1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and
- is able to cleave the aminoacylatable 3' tRNA-like structure (TLS) of a plant RNA
virus.
[0008] A protein-only RNase P enzyme (PRORP, for PROtein-only RNase P) is a protein with
RNase P activity composed of an N-terminal α-super helix domain containing PPR motifs
and a C-terminal NYN-type metallonuclease domain connected by a zinc-binding module
(see Figure 1, Gobert
et al., 2010 and 2013; Gutmann
et al., 2012; Howard
et al., 2012).
N-terminal α-super helix domain containing PPR motifs
[0009] PPR domains contain 35 amino-acid repeats involved in RNA binding. They are eukaryote-specific.
The primary sequence of PPR repeats is highly degenerate but their 3D structure is
conserved. Each repeat is composed of an α-helix-tum-helix structure (Lurin
et al., 2004). Structural predictions of the N-terminal domains of PRORP enzymes from diverse
organisms using Phyre2 (Kelley and Sternberg, 2009) are congruent with the occurrence
of a conserved α-super helix corresponding to the fold of PPR proteins (Small and
Peeters, 2000). Predicted numbers of PPR repeats in PRORP sequences from various organisms
range from 2 to 5 according to the TPRpred prediction software (Karpenahalli
et al., 2007). As inferred from footprinting data (Gobert
et al., 2013) and protein truncation (Howard
et al., 2012), the N-terminal part of PRORP bears the RNA recognition domain that binds conserved
nucleotides and structure elements in the D- and TψC-loops of tRNAs and tRNA-like
structures (Gobert
et al., 2013).
C-terminal NYN-type metallonuclease domain
[0010] The second main domain of PRORP enzymes is a metallonuclease domain (see Figure 1)
responsible for the actual nuclease activity of these enzymes (Gobert
et al., 2010; Gutmann
et al., 2012; Howard
et al., 2012). This catalytic domain is a PIN-like domain from the NYN family. The NYN catalytic
domain seems to originate from the putative bacterial ribonuclease coded by the
yacP gene and is found throughout the main branches of life, with the exception of fungi
(Anantharaman and Aravind, 2006). This domain contains conserved aspartate residues,
but additional conserved residues are characteristic for the PRORP subfamily and allow
to discriminate between PRORP and other NYN-domain proteins. The NYN domain of PRORP
is characterized by 2 subparts. The first part comprises the following signature (D/E/T/H/N/P/G)h
3D(G/A)xN (SEQ ID NO: 1). In this subpart of the signature, "h
3" correspond to hydrophobic amino acids with few exceptions where 1 out of 3 amino
acids is nucleophylic. The first acidic amino acid (D/E) is highly conserved in land
plants. The second subpart of the domain contains the following conserved signature
DDx
15(S/T)xDx
3DH (SEQ ID NO: 2). The length between the first and the second sub-domain is restricted
to 70-80 amino acids in land plants.
Connecting zinc-binding module
[0011] The PPR and NYN domains are connected by a zinc-binding module composed of 2 subparts
placed upstream and downstream of the NYN domain (see Figure 1). The first part contains
a CxxC (SEQ ID NO: 3) motif strictly conserved in all PRORP sequences of land plants.
This signature is found between 49 and 144 amino acid upstream of the NYN domain.
The second subpart contains the following signatures (W/Y/F)HxPx (SEQ ID NO: 4), and
(W/F)xCx
2-3(R/K) (SEQ ID NO: 5). The conserved C and H residues of this bi-partite module are
implicated in zinc binding (Howard
et al., 2012; Gobert
et al., 2013).
Other conserved motifs of PRORP
[0012] Further signatures are presents in specific phyla. In land plants, a stretch of three
to four glycines (Gs) separates the protein between the two main domains just before
the zinc-binding module (see Figure 1). Charged residues upstream of the Gs face the
outside of PRORP structure and it might be involved in protein/protein interactions.
In addition, a motif generally composed of MPxP(Y/F/C)(S/T) (SEQ ID NO: 6) is present
between the NYN domain and the C-terminal part of the zinc-binding module (see Figure
1).
PRORP MTS and NLS domains
[0013] PRORP MTS sequences are N-terminal amphiphilic α-helices structures. Their occurence
can be predicted by softwares such as Predotar (Small
et al., 2004) or TargetP (Emanuelsson
et al., 2000). PRORP NLS sequences are mono or bi-partite sequences located most of the times
in the N-terminal and/or C-terminal parts of proteins, characterized by the frequent
occurrence of basic residues such as Lysines and Arginines. Their occurence can be
predicted by softwares such as NLStradamus (Nguyen Ba
et al., 2009).
Numbering of important positions in Arabidopsis PRORP2 (SEQ ID NO: 105)
[0014]
- The nuclear localization signal of AtPRORP2 is composed of residues 1 to 24.
- Among the 5 PPR repeats found in AtPRORP2, the positions L34, S65, Q70, N108, S114,
R145, A150, E180, S185, S215 are predicted to be important for RNA substrate recognition,
according to the proposed PPR / RNA recognition code (Barkan et al., 2012).
- The bi-partite zinc-binding motif contains residues C281 and C284 in the middle of
AtPRORP2 sequence and residues W493, H494, P496, C511 and R514 at the C-terminal end
of AtPRORP2 sequence.
- In the conserved NYN domain, residues E339, D343, D421, D422, D440, D444 and H445
are predicted to be of high functional importance. Residues D421 and D422 were already
shown to be essential for catalytic activity (Gutmann et al., 2012).
- Other motifs are characteristic of plant AtPRORP enzymes. Among them, a motif composed
by G254, G255 and G256 is present in AtPRORP2. Another motif of unknown function contains
the conserved M477 and S482.
Methods for retrieving PRORPs
[0015] PRORP sequences can be retrieved using the BLAST tool (Altschul
et al., 1990) in the following databases:
NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi),
Ensembl (http://www.ensembl.org/Multi/blastview),
Bogas (http://bioinformatics.psb.ugent.be/webtools/bogas/),
Phytozome (http://www.phytozome.net/),
JGI (http://genome.jgi-psf.org/) and/or
Broad (http://www.broadinstitute.org/scientific-community/data).
[0016] The sequences can be aligned using Muscle (EMBL-EBI: http://www.ebi.ac.uk/Tools/msa/muscle/)
(Edgar, 2004) before using WebLogo 3 ((Crooks
et al., 2004), http://weblogo.threeplusone.com/create.cgi) to highlight the conserved residues.
[0017] Advantageously, the protein-only RNase P enzyme contains the residues G254, G255,
G256, C281, C284, E339, D343, D421, D422, D440, D444, H445, M477, S482, W493, H494,
P496, C511 and R514, and eventually at least one of the residues selected from the
group consisting of L34, S65, Q70, N108, S114, R145, A150, E180, S185 and S215, numbered
according to
A. thaliana PRORP2 sequence represented as SEQ ID NO: 105.
[0018] Advantageously, the protein-only RNase P enzyme has the consensus amino acid sequence
described in Figure 2 (SEQ ID NO: 7).
[0019] Advantageously, the protein-only RNase P enzyme is from a land plant, such as a PRORP
enzyme selected from the group consisting of
Oryza sativa (rice) SEQ ID NO: 8, 9 or 10;
Zea mays (corn) SEQ ID NO: 11, 12 or 13;
Triticum turgidum (wheat) SEQ ID NO: 14 or 15;
Solanum lycopersicum (tomato) SEQ ID NO: 16, 17 or 18;
Brassica rapa (turnip) SEQ ID NO: 19, 20 or 21;
Carica papaya (papaya) SEQ ID NO: 22, 23 or 24;
Solanum tuberosum (potato) SEQ ID NO: 25, 26 or 27;
Nicotiana tabacum (tobacco) SEQ ID NO: 28, 29 or 30;
Setaria (millet) SEQ ID NO: 31, 32 or 33;
Sorghum bicolor (sorghum) SEQ ID NO: 34, 35, 36 or 37;
Hordeum vulgare (barley) SEQ ID NO: 38 or 39;
Oryza officinalis (rice) SEQ ID NO: 40;
Manihot esculenta (manioc) SEQ ID NO: 41, 42 or 43;
Theobroma cacao (cocoa) SEQ ID NO: 44, 45 or 46;
Cucumis sativus (cucumber) SEQ ID NO: 47, 48 or 49;
Vitis vinifera (vine) SEQ ID NO: 50, 51, 52 or 53;
Glycine max (soybean) SEQ ID NO: 54, 55, 56, 57 or 58;
Prunus persica (peach) SEQ ID NO: 59, 60 or 61;
Malus domestica (apple) SEQ ID NO: 62, 63 or 64;
Fragaria vesca (strawberry) SEQ ID NO: 65, 66 or 67;
Citrus clementina (clementine) SEQ ID NO: 68, 69 or 70;
Citrus sinensis (orange) SEQ ID NO: 71, 72 or 73;
Populus trichocarpa (poplar) SEQ ID NO: 74, 75, 76, 77 or 78;
Eucalyptus SEQ ID NO: 79, 80 or 81;
Ricinus communis (ricinus) SEQ ID NO: 82, 83 or 84;
Medicago sativa (alfalfa / lucerne) SEQ ID NO: 85, 86 or 87;
Lotus SEQ ID NO: 88 or 89;
Aquilegia (columbine) SEQ ID NO: 90, 91 or 92;
Eutrema halophila SEQ ID NO: 93, 94 or 95;
Eutrema parvulum SEQ ID NO: 96, 97 or 98;
Mimulus (monkey-flower) SEQ ID NO: 99, 100, 101 or 102;
Jatropha SEQ ID NO: 103;
Arabidopsis thaliana SEQ ID NO: 104, 105 or 106;
Arabidopsis lyrata SEQ ID NO: 107, 108 or 109;
Brachypodium SEQ ID NO: 110, 111 or 112;
Physcomitrella patens SEQ ID NO: 113, 114 or 115;
Selaginella moellendorffii SEQ ID NO: 116 or 117; and a PRORP enzyme comprising the
Brassica napus (rapeseed) sequence SEQ ID NO: 118, 119 or 120.
[0020] According to a preferred embodiment of the invention, the mutant protein-only RNase
P enzyme (CytoRP) is able to cleave the aminoacylatable 3' tRNA-like structure (TLS)
of a plant RNA virus belonging to a genus selected from the group consisting of Tymovirus,
Furovirus, Pomovirus, Pecluvirus, Tobamovirus, Bromovirus, Cucumovirus and Hordeivirus.
More preferably CytoRP is able to cleave the aminoacylatable 3' TLS of a plant RNA
virus selected from the group consisting of Turnip yellow mosaic virus (TYMV), Andean
potato latent virus (APLV), Belladonna mottle virus (BeMV), Cacao yellow mosaic virus
(CYMV), Clitoria yellow vein virus (CYVV), Eggplant mosaic virus (EMV), Kennedya yellow
mosaic virus (KYMV), Okra mosaic virus (OkMV), Ononis yellow mosaic virus (OYMV),
Wild cucumber mosaic virus (WCMV), Nemesia ring necrosis virus (NeRNV), Soil-borne
wheat mosaic virus (SBWMV), Beet soil-borne virus (BSBV), Potato mop-top virus (PMTV),
Indian peanut clump virus (IPCV), Peanut clump virus (PCV), Tobacco mosaic virus (TMV),
Cucumber green mottle mosaic virus (CGMMV), Green tomato atypical mosaic virus (GTAMV),
Satellite tobacco mosaic virus (STMV), Sunnhemp mosaic virus (SHMV), Brome mosaic
virus (BMV), Broad bean mottle virus (BBMV), Cowpea chlorotic mottle virus (CCMV),
Cucumber mosaic virus (CMV), Barley stripe mosaic virus (BSMV) and Poa semilatent
virus (PSLV). More preferably CytoRP is able to cleave the aminoacylatable 3' TLS
of a plant RNA virus selected from the group consisting of TYMV, TMV and BMV.
[0021] The aminoacylatable 3' TLS of the above-mentioned plant RNA viruses is known in the
art (see for review Dreher, 2010).
[0022] By way of example, the nucleotide sequence of the aminoacylatable 3' TLS of the TYMV
is represented as SEQ ID NO: 237, of the TMV is represented as SEQ ID NO: 238, and
of the BMV is represented as SEQ ID NO: 239.
[0023] The cleavage of the aminoacylatable 3' tRNA-like structure (TLS) of a plant RNA virus
by a mutant protein-only RNase P enzyme (CytoRP) according to the present invention
can be determined
in vitro. In vitro cleavage assays are described in the Examples below, in Gobert
et al., 2010 and in Gutmann
et al., 2012.
[0024] According to another preferred embodiment of the invention, the mutant protein-only
RNase P enzyme (CytoRP) has at least 50% identity, or by order of increasing preference
at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity with a polypeptide of sequence selected from the group consisting
of SEQ ID NO: 121, 122 or 123 (mutants of PRORP enzymes from
Oryza sativa wherein the NLS or MTS domain is deleted); SEQ ID NO: 124, 125 or 126 (mutants of
PRORP enzymes from
Zea mays wherein the NLS or MTS domain is deleted); SEQ ID NO: 127 or 128 (mutants of PRORP
enzymes from
Triticum turgidum); SEQ ID NO: 129, 130 or 131 (mutants of PRORP enzymes from
Solanum lycopersicum wherein the NLS or MTS domain is deleted); SEQ ID NO: 132, 133 or 134 (mutants of
PRORP enzymes from
Brassica rapa wherein the NLS or MTS domain is deleted); SEQ ID NO: 135, 136 or 137 (mutants of
PRORP enzymes from
Carica papaya wherein the NLS or MTS domain is deleted); SEQ ID NO: 138, 139 or 140 (mutants of
PRORP enzymes from
Solanum tuberosum wherein the NLS or MTS domain is deleted); SEQ ID NO: 141, 142 or 143 (mutants of
PRORP enzymes from
Nicotiana tabacum wherein the NLS or MTS domain is deleted); SEQ ID NO: 144, 145 or 146 (mutants of
PRORP enzymes from
Setaria wherein the NLS domain is deleted); SEQ ID NO: 147, 148, 149 or 150 (mutants of PRORP
enzymes from
Sorghum bicolor wherein the NLS domain is deleted); SEQ ID NO: 151 or 152 (mutants of PRORP enzymes
from
Hordeum vulgare wherein the NLS or MTS domain is deleted); SEQ ID NO: 153 (mutant of PRORP enzyme
from
Oryza officinalis wherein the NLS domain is deleted); SEQ ID NO: 154, 155 or 156 (mutants of PRORP
enzymes from
Manihot esculenta wherein the NLS or MTS domain is deleted); SEQ ID NO: 157, 158 or 159 (mutants of
PRORP enzymes from
Theobroma cacao wherein the NLS or MTS domain is deleted); SEQ ID NO: 160, 161 or 162 (mutants of
PRORP enzymes from
Cucumis sativus wherein the NLS or MTS domain is deleted); SEQ ID NO: 163, 164, 165 or 166 (mutants
of PRORP enzymes from
Vitis vinifera wherein the NLS or MTS domain is deleted); SEQ ID NO: 167, 168, 169, 170 or 171 (mutants
of PRORP enzymes from
Glycine max wherein the NLS or MTS domain is deleted); SEQ ID NO: 172, 173 or 174 (mutants of
PRORP enzymes from
Prunus persica wherein the NLS or MTS domain is deleted); SEQ ID NO: 175, 176 or 177 (mutants of
PRORP enzymes from
Malus domestica wherein the NLS or MTS domain is deleted); SEQ ID NO: 178, 179 or 180 (mutants of
PRORP enzymes from
Fragaria vesca wherein the NLS or MTS domain is deleted); SEQ ID NO: 181, 182 or 183 (mutants of
PRORP enzymes from
Citrus clementina wherein the NLS domain is deleted); SEQ ID NO: 184, 185 or 186 (mutants of PRORP
enzymes from
Citrus sinensis wherein the NLS domain is deleted); SEQ ID NO: 187, 188, 189, 190 or 191 (mutants
of PRORP enzymes from
Populus trichocarpa wherein the NLS or MTS domain is deleted); SEQ ID NO: 192, 193 or 194 (mutants of
PRORP enzymes from
Eucalyptus wherein the NLS or MTS domain is deleted); SEQ ID NO: 195, 196 or 197 (mutants of
PRORP enzymes from
Ricinus communis wherein the NLS or MTS domain is deleted); SEQ ID NO: 198, 199 or 200 (mutants of
PRORP enzymes from
Medicago sativa wherein the NLS or MTS domain is deleted); SEQ ID NO: 201 or 202 (mutants of PRORP
enzymes from
Lotus wherein the MTS domain is deleted); SEQ ID NO: 203, 204 or 205 (mutants of PRORP
enzymes from
Aquilegia wherein the NLS or MTS domain is deleted); SEQ ID NO: 206, 207 or 208 (mutants of
PRORP enzymes from
Eutrema halophila wherein the NLS or MTS domain is deleted); SEQ ID NO: 209, 210 or 211 (mutants of
PRORP enzymes from
Eutrema parvulum wherein the NLS or MTS domain is deleted); SEQ ID NO: 212, 213, 214 or 215 (mutants
of PRORP enzymes from
Mimulus wherein the NLS or MTS domain is deleted); SEQ ID NO: 216 (mutant of PRORP enzyme
from
Jatropha wherein the NLS domain is deleted); SEQ ID NO: 217, 218 or 219 (mutants of PRORP
enzymes from
Arabidopsis thaliana wherein the NLS or MTS domain is deleted); SEQ ID NO: 220, 221 or 222 (mutants of
PRORP enzymes from
Arabidopsis lyrata wherein the NLS or MTS domain is deleted); SEQ ID NO: 223, 224 or 225 (mutants of
PRORP enzymes from
Brachypodium wherein the NLS or MTS domain is deleted); SEQ ID NO: 226, 227 or 228 (mutants of
PRORP enzymes from
Physcomitrella patens wherein the NLS or MTS domain is deleted); and SEQ ID NO: 229 or 230 (mutants of
PRORP enzymes from
Selaginella moellendorffii wherein the NLS or MTS domain is deleted).
[0025] Unless otherwise specified, the protein sequence identity values provided herein
are calculated using the BLASTP program under default parameters, on a comparison
window including the whole sequence of the proteins to be compared.
[0026] Advantageously, CytoRP consists in an amino acid sequence selected from the group
consisting of SEQ ID NO: 121 to 230.
[0027] According to another preferred embodiment of the invention, CytoRP is a mutant of
an endogenous protein-only RNase P enzyme from said plant to which the method of the
invention is applied.
[0028] According to another preferred embodiment of the invention, the method is for increasing
the resistance of a plant to a plant RNA virus belonging to a genus selected from
the group consisting of Tymovirus, Furovirus, Pomovirus, Pecluvirus, Tobamovirus,
Bromovirus, Cucumovirus and Hordeivirus. In particular, said plant RNA virus is selected
from the group consisting of Turnip yellow mosaic virus (TYMV), Andean potato latent
virus (APLV), Belladonna mottle virus (BeMV), Cacao yellow mosaic virus (CYMV), Clitoria
yellow vein virus (CYVV), Eggplant mosaic virus (EMV), Kennedya yellow mosaic virus
(KYMV), Okra mosaic virus (OkMV), Ononis yellow mosaic virus (OYMV), Wild cucumber
mosaic virus (WCMV), Nemesia ring necrosis virus (NeRNV), Soil-borne wheat mosaic
virus (SBWMV), Beet soil-borne virus (BSBV), Potato mop-top virus (PMTV), Indian peanut
clump virus (IPCV), Peanut clump virus (PCV), Tobacco mosaic virus (TMV), Cucumber
green mottle mosaic virus (CGMMV), Green tomato atypical mosaic virus (GTAMV), Satellite
tobacco mosaic virus (STMV), Sunnhemp mosaic virus (SHMV), Brome mosaic virus (BMV),
Broad bean mottle virus (BBMV), Cowpea chlorotic mottle virus (CCMV), Cucumber mosaic
virus (CMV), Barley stripe mosaic virus (BSMV), Poa semilatent virus (PSLV).
[0029] The term "plant" includes any monocotyledon or dicotyledon plant.
[0030] Advantageously, the invention applies to plants of agronomical interest, such as
rice, corn, wheat, tomato, turnip, papaya, rapeseed, potato, tobacco, millet, sorghum,
barley, manioc, cocoa, cucumber, vine, soybean, peach, apple, strawberry, clementine,
orange, poplar, eucalyptus, ricinus, alfalfa (lucerne), lotus, carrot, pepper, aubergine,
zucchini, melon, bean, spinach, lettuce, onion, celery, beet, squash and strawberry,
preferably potato, potato, cucumber, tobacco, carrot, pepper, aubergine, zucchini,
melon, bean, spinach, lettuce, celery, beet, squash and strawberry, more preferably
tobacco, cucumber, tomato, lettuce and onion.
[0031] A preferred method for expressing a mutant protein-only RNase P enzyme (CytoRP) according
to the present invention comprises introducing into the genome of said plant a DNA
construct comprising a nucleotide sequence encoding said CytoRP, placed under control
of a promoter.
[0032] The instant invention also provides means for expressing a mutant protein-only RNase
P enzyme (CytoRP).
[0033] This included an isolated polynucleotide encoding a CytoRP as defined above.
[0034] This also includes recombinant DNA constructs for expressing a CytoRP enzyme in a
host-cell (e.g., bacteria or plant cell) or a whole plant. These recombinant DNA constructs
can be obtained and introduced in said host cell or whole plant by well known techniques
of recombinant DNA and genetic engineering.
[0035] Recombinant DNA constructs of the invention include expression cassettes, comprising
a polynucleotide encoding a CytoRP as defined above, under control of a transcription
promoter functional in a host cell (
e.g., bacteria or plant cell).
[0036] Said transcription promoter may be any promoter that is functional in a cell, preferably
a plant cell,
i.e., capable of directing the transcription of a polynucleotide encoding a CytoRP as defined
above in a cell, preferably a plant cell (for review, see Yoshida and Shinmyo, 2000).
The choice of the most appropriate promoter depends in particular on the organ(s)
or on the tissue(s) targeted for the expression. The promoter may be a constitutive
promoter (
i.e., a promoter which is active in most tissues and cells and under most environmental
conditions), a cell-type-specific promoter (
i.e., a promoter which is active only or mainly in certain tissues or certain types of
cells) or an inducible promoter (
i.e., a promoter which is activated by physical processes or chemical stimuli). The promoter
may also be the promoter of a
PRORP gene, such as the A.
thaliana PRORP2 promoter included in SEQ ID NO: 231.
[0037] By way of non-limiting examples of constitutive promoters which are commonly used
in plant cells, mention may be made of the cauliflower mosaic virus (CaMV) 35S promoter,
the NOS (nopaline synthase) promoter, the PG10-90 synthetic promoter, preferably the
35S promoter.
[0038] By way of non-limiting examples of organ-specific or tissue-specific promoters, mention
may be made of promoters such as the pollen specific APRS promoter, the embryo specific
MXL promoter (Jopcik
et al., 2013) or any plant promoter as listed in the plant promoter database PlantProm (Shahmuradov
et al., 2003).
[0039] By way of non-limiting examples of inducible promoters, mention may be made of the
ethanol inducible AlcR/AlcA and the β-estradiol inducible XVE/OlexA inducible systems
(Borghi, 2010).
[0040] Said recombinant expression cassette may also comprise a transcription terminator,
such as, for example, the CaMV 35S terminator, the NOS terminator or the T9 terminator
of the rbcS E9 gene. The terminator may also be the terminator of a
PRORP gene, such as the A.
thaliana PRORP2 terminator included in SEQ ID NO: 232.
[0041] Said recombinant expression cassette may also include other regulatory sequences,
such as transcription enhancer sequences.
[0042] Recombinant DNA constructs of the invention also include recombinant vectors containing
an expression cassette comprising a polynucleotide encoding a CytoRP as defined above.
In particular said expression cassette is a recombinant expression cassette of the
invention, wherein the polynucleotide encoding a CytoRP is under control of a promoter
of a
PRORP gene.
[0043] The expression cassettes and the expression vectors according to the invention may
also comprise other sequences, usually employed in constructs of this type, such as
translation leader (TL) sequences, polyadenylation sites, and also, where appropriate,
amplifying sequences (transcription enhancer sequences). They may also comprise sequences
which make it possible to monitor the transformation, and also to identify and/or
to select the cells or organisms transformed. These are, in particular, reporter genes
(for example the
beta-glucuronidase (GUS) gene, the
luciferase gene or the
green fluorescent protein (GFP) gene, conferring an easily recognizable phenotype on these cells or organisms,
or else selection marker genes (for example, genes for resistance to an antibiotic,
such as kanamycin or hygromycin, or to an herbicide).
[0044] The choice of the promoter and of the additional sequences that can be inserted into
the expression cassettes and vectors according to the invention can be made, conventionally,
by a person skilled in the art according in particular to criteria such as the host
vector, host cells and organisms chosen, the desired expression profile in the host
cell or organism, the genetic transformation protocols envisioned, etc.
[0045] The selection of suitable vectors and the methods for inserting DNA constructs therein
are well known to a person skilled in the art. The choice of the vector depends on
the intended host and on the intended method of transformation of said host.
[0046] A variety of techniques for genetic transformation of plant cells or plants are available
in the art. By way of non-limiting examples, one can mention methods of direct transfer
of genes such as direct micro-injection into plant embryoids, vacuum infiltration
or electroporation, or the bombardment by gun of particules covered with the plasmidic
DNA of interest.
Agrobacterium mediated transformation methods may also be used such as
Agrobacterium tumefaciens or
Agrobacterium rhizogenes.
[0047] The present invention also provides a host cell comprising an expression cassette
or a recombinant vector as defined above.
[0048] The host cells can be prokaryotic or eukaryotic cells. In the case of prokaryotic
cells, they may be agrobacteria such as
Agrobacterium tumefaciens or
Agrobacterium rhizobium. In the case of eukaryotic cells, they may be plant cells stemming from dicotyledonous
or monocotyledonous plants.
[0049] The invention also provides a method for producing a transgenic plant, having an
increased resistance to a plant RNA virus.
[0050] Various methods for obtaining transgenic plants are well known in themselves to a
person skilled in the art.
[0051] In particular, said method comprises transforming a plant cell by a DNA construct
of the invention and regenerating from said plant cell a transgenic plant expressing
a CytoRP as defined above.
[0052] According to a preferred embodiment of this, it comprises transforming a plant cell
with a recombinant vector of the invention comprising a polynucleotide encoding a
CytoRP as defined above, and regenerating from said plant cell a transgenic plant
expressing a CytoRP.
[0053] A very large number of techniques for transforming plant germinal or somatic cells
(isolated, in the form of tissue or organ cultures, or on the whole plant), and regenerating
the plants are available. The choice of the most suitable method generally depends
on the plant concerned.
[0054] The invention also comprises plants genetically transformed by a recombinant DNA
construct of the invention, such as an expression cassette, and expressing a CytoRP
as defined above, and in particular transgenic plants comprising, in their nuclear
genome, at least one copy of a transgene containing a recombinant DNA construct of
the invention according to the invention. In said transgenic plants a DNA construct
of the invention is comprised in a transgene stably integrated in the plant genome,
so that it is passed onto successive plant generations. Thus the transgenic plants
of the invention include not only the plants resulting from the initial transgenesis,
but also their descendants, as far as they contain a recombinant DNA construct of
the invention. The expression of a CytoRP as defined above in said plants provides
them an increased resistance to a plant RNA virus, when compared with a wild-type
plant devoid of said transgene(s).
[0055] The disclosure also discloses a transgenic plant, obtainable by a method of the invention,
expressing a CytoRP as defined above, said plant containing a recombinant expression
cassette of the invention.
[0056] The invention further comprises a transgenic plant or an isolated organ or tissue
thereof (such as seeds, leafs, flowers, roots, stems, ears) comprising, stably integrated
in its genome, a recombinant expression cassette comprising a polynucleotide encoding
a CytoRP as defined above.
[0057] The present disclosure also discloses an isolated mutant protein-only RNase P enzyme
(CytoRP) comprising neither a nuclear localization signal (NLS) domain nor an organelle
targeting sequence (MTS) domain, and able to cleave the aminoacylatable 3' tRNA-like
structure (TLS) of a plant RNA virus, as defined above.
[0058] The present invention also provides a protein which is a CytoRP according to the
invention.
[0059] Advantageously, CytoRP has at least 50% identity, or by order of increasing preference
at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identity with a polypeptide of sequence selected from the group consisting
of SEQ ID NO: 121 to 230.
[0060] The present invention also provides the use of an isolated polynucleotide encoding
a CyroRP as defined above for producing a transgenic plant having an increased resistance
to a plant RNA virus as defined above.
[0061] Foregoing and other objects and advantages of the invention will become more apparent
from the following detailed description and accompanying drawing, which refers to
non-limiting examples illustrating the use of a CytoRP for increasing the resistance
of a plant to a plant RNA virus. It is to be understood however that this foregoing
detailed description is exemplary only and is not restrictive of the invention.
Figure 1: Features defining PRORP enzymes. Schematic representation of a PRORP enzyme with
residues predicted to play an important role highlighted and numbered according to
Arabidopsis PRORP2 sequence.
Figure 2 (A-I) Schematic representation of residue frequency at each position in an alignment of
91 plant PRORP sequences, representing the consensus amino acid sequence of PRORP.
The alignment has been obtained with the software LogoBar (Pérez-Bercoff et al., 2006; http://www.biosci.ki.se/groups/tbu/logobar).
Figure 3: The fusion of CytoRP with eYFP observed by confocal microscopy shows the cytosolic
localization of CytoRP. This location differs from the nuclear localization of PRORP2/3
and the organellar localization of PRORP1 (Gobert et al., 2010). "C" shows location controls with the auto-fluorescence of chlorophyll revealing
chloroplasts and DAPI staining (for PRORP2/3) revealing DNA in the nucleus. "T" shows
cells in transmitted light.
Figure 4: Cytosolic localization of CytoRP (mutant AtPRORP2 wherein the NLS domain is deleted)
determined by laser scanning confocal microscopy. Four representative experiments
are shown where the construct expressing CytoRP-eYFP fusion was transformed into Arabidopsis protoplasts. In each experiment, the respective panels show for each cell, the eYFP
signal, the autofluorescence of chlorophyll showing chloroplasts, the transmitted
light image of cells and the merged image of the eYFP and the autofluorescence channels.
Figure 5: Transformed plants encoding CytoRP (mutant AtPRORP2 wherein the NLS domain is deleted)
were identified by PCR using total genomic DNA from plant extracts and oligonucleotides
P2-5'UTR-F (SEQ ID NO: 135) and P2-R (SEQ ID NO: 136) present in AtPRORP2 5' UTR region
and in its coding region respectively. Lanes 1, 2, 3 and 4 correspond to PCR reactions
performed on plant genomic DNA. Lanes 5 and 6 correspond to PCR reactions performed
on cDNA clones representing PRORP2 and PRORP2ΔNLS (CytoRP) respectively, serving as
PCR positive controls and size references. M shows the molecular weight marker. DNA
fragments were separated on a 1% agarose gel, stained with ethidium bromide and visualised
under UV light. 1, 2 and 3 correspond to wild type plants that encode PRORP2, whereas
4, highlighted by an asterisk, corresponds to a CytoRP plant that expresses PRORP2ΔNLS.
Figure 6: RNase P activity assays of Arabidopsis CytoRP on in vitro synthesized transcripts representing tRNA like structures of plant viruses TYMV,
TMV and BMV. (-) indicate lanes with TLS transcripts alone and (+) indicate lanes
where transcripts were incubated with CytoRP proteins resulting in specific cleavage
patterns.
Figure7: PCR sample of 2 µl (from the 50 µl) mixed with water (3 µl) and 6x DNA loading dye
(1 µl) and charged into a 1 % agarose gel. 4.5 µl MassRuler (Thermo Scientific) was
used for size determination. P = PRORP.
Figure 8: 1% agarose gel is prepared and 5 µl PCR product was loaded. 4.5 µl Mass-Ruler was
added for size determination. (A) Gel 1 shows for N. tabacum PRORP1 that colonies 1, 2 and 6 have the right size amplification for NtPRORP1, for
N. tabacum PRORP2 that none of the colonies have NtPRORP2. (B) Gel 2 shows for N. tabacum PRORP3 that colony 1 has the right size amplification for NtPRORP3, for C. sativus PRORP3 that colonies 1, 2, 3, 5, 6 and 8 have the right size amplification for CsPRORP3.
(C) Gel 3 shows for A. cepa PRORP3 that colonies 2, 3, 4, 5, 6, 8, 10, 11, 12 have about the right size amplification
for CsPRORP3, for L. sativa PRORP3 that colonies 3 and 7 have the right size amplification for LSPRORP3, for
a new transformation of pGEM-T easy:N. tabacumPRORP3 in E. coli TOP10 that colonies 3, 5, 6, 8, 9, 11, 12 have the right size amplification for NtPRORP3,
for S. lycopersicum PRORP3 that colonies 1, 4, 6, 7, 8, 10 and 12 have the right size amplification for
S1PRORP3.
EXAMPLE I: EXPERIMENTAL VALIDATION OF THE USE OF CYTORP FOR INCREASING THE RESISTANCE
OF A PLANT TO A PLANT RNA VIRUS
1. Methods
CytoRP enzyme
[0062] The CytoRP protein (SEQ ID NO: 218), whose construction is described here, derives
from the
Arabidopsis PRORP2 protein (At2g16650). Its coding sequence corresponds to nucleotides 73 to
1587 of the native PRORP2 cDNA, preceded by an ATG initiation codon. CytoRP is the
result of the deletion of the first 24 amino acids of PRORP2 (corresponding to the
NLS domain). CytoRP is a protein of 505 amino acids with a molecular weight of 56539,73
Da with an isoelectric point of 7.06.
Localization experiments
[0063] CytoRP cDNA (SEQ ID NO: 233) was inserted into the pART7eYFP vector (Gleave, 1992).
Arabidopsis mesophyll protoplasts were isolated and transformed with pART7CytoRPeYFP plasmids
as described previously (Abel and Theologis, 1994).
[0064] Briefly, plant material was put in Plasmolysis solution containing 0.4 M mannitol,
3% sucrose and 8 mM CaCl
2 and incubated 30 min at room temperature (RT). Cells were spun at 42 g for 10 min
at RT, resuspended in Enzyme solution containing 1% (w/v) cellulase and 0.25% (w/v)
macerozyme diluted in Plasmolysis solution and incubated in the dark at RT for 1 h
and for a further 1 to 2 h on an orbital shaker at 20 rpm. The obtained protoplasts
were filtered through a nylon sieve (100 µm), washed by adding 30 ml of 0.4 M mannitol
in W5 solution (5 mM glucose, 154 mM NaCl, 125 mM CaCl
2, 5 mM KCl and 1.5 mM MES pH 5.6) and spun at 42 g for 10 min at RT. Protoplasts were
then washed twice by adding 10 ml of mannitol/Mg solution (0.4 M mannitol, 0.1% MES
and 15 mM MgCl
2) and finally re-suspended in 10 ml of mannitol/Mg solution.
[0065] To transform protoplasts, 50 µg of plasmid DNA were mixed to 250 µg of herring sperm
carrier DNA, cleaned by three cycles of phenol/chloroform extractions and ethanol
precipitations, re-suspended in 50 µl of H
2O and mixed with 25 µl of chloroform. This solution was deposited in droplets on a
petri dish next to 300 µl of protoplast solution (2.10
6 protoplasts) and 350 µl of PEG. The protoplasts were mixed to the DNA by gentle swirls
to the plate. The PEG was injected at once by fusing the drop of DNA/protoplasts to
the drop of PEG. Protoplasts were then diluted with 600 µl, 1 ml, 2 ml and 4 ml of
0.4 M mannitol in W5 solution added in droplets every 3 min. The diluted protoplasts
were harvested by spinning at 20 g for 5 min at RT. Transformed protoplasts were re-suspended
in 2 ml of culture medium (0.4 M sucrose, 1x Murashige and Skoog basal media and 250
mg/l xylose) and cultivated in the dark at 20°C for 48 h.
[0066] Transformed protoplasts were visualised by confocal microscopy. eYFP fluorescence
was observed by confocal laser scanning microscopy using a Zeiss LSM510 based on an
Axiovert 200M microscope (Zeiss).
Arabidopsis stable transformation
[0067] A PCR amplified DNA fragment (SEQ ID NO: 234) containing CytoRP cDNA sequence (SEQ
ID NO: 233) as well as AtPRORP2 promoter (SEQ ID NO: 231) and terminator (SEQ ID NO:
232) sequences was cloned in the binary vector pGWB13 (Nakagawa
et al., 2007). The construct obtained was used to transform
Arabidopsis thaliana ecotype col0 plants by the "floral dip" method (Clough and Bent, 1998).
[0068] Briefly, the pGWB13 construct, carrying the CyoRP insert as well as a hygromycin
resistance marker gene, was transformed in
Agrobacterium tumefaciens strain GV3101 cells. Bacteria were grown at 28°C in liquid LB medium until OD
600 = 0.8, centrifuged and resuspended in 5% Sucrose and 0.05% Silwet L-77 solution.
Aerial parts of
Arabidopsis plants were dipped 3 times in the
Agobacterium solution for 3 seconds with gentle agitation. Dipped plants were then placed under
cover for 24 hours to maintain high humidity and grown according to standard conditions.
Dry seeds were harvested and next generation individual plants analysed to test for
their resistance to hygromycin and thus to identify individual transformants.
RNase P activity assays
Production of recombinant PRORP enzymes
[0069] PRORP cDNAs were cloned in pET28-b(+) (Novagen) to obtain C terminal fusions with histidine
affinity tags. Proteins were expressed over night at 18°C in BL21(DE3)
E.coli cells induced with 1 mM IPTG. Bacteria were lysed and centrifuged 30 min at 30,000
rpm 4°C. The cleared bacterial lysates were incubated with the Ni NTA resin (Qiagen).
The bound proteins were washed with buffers containing 50 mM imidazole, 20 mM MOPS
pH 7.8, 150 mM NaCl and 15% (v/v) glycerol and 75 mM imidazole, 20 mM MOPS pH 7.8,
250 mM NaCl and 15% (v/v) glycerol. Proteins were eluted from the column using 200
mM imidazole and 500 mM imidazole buffers.
Production of transcripts representing tRNA like structures
[0070] cDNAs representing TLS containing RNAs were amplified by PCR using 5' oligonucleotides
containing T7 promoter sequences. PCR products were cloned in pUC19.
[0071] 200 ng of linearized plasmid DNA were transcribed in a volume of 10 µl containing
7.5 mM rNTP, 5 U T7 RNA polymerase and buffer as supplied by the manufacturer (RiboMAX,
Promega) for 4 h at 37°C. After this, plasmid DNA was digested with 1 U of RQ1 DNase
for 15 min at 37°C and synthesized RNA were purified by phenol chloroform extractions.
Transcripts were dephosphorylated with 1 U FastAP Alkaline Phosphatase (Fermentas)
for 30 min at 37°C and 5' radiolabelled with
32P-γATP and polynucleotide kinase (Fermentas).
Cleavage assays
[0072] Reactions were performed essentially as described previously (Gobert
et al., 2010) with proteins purified as described (Gobert
et al., 2010). Reactions were performed in 10 µl with 100 ng proteins and 100 ng 5' radiolabelled
RNA, in buffer containing 20 mM Tris-HCl pH 8, 30 mM KCl, 4.5 mM MgCl
2, 20 µg/ml BSA and 2 mM DTT, for 15 min at room temperature. RNA molecules were separated
on 8% polyacrylamide urea gels and visualized by ethidium bromide staining and / or
by autoradiography.
Validation of the antiviral strategy
[0073] The degree of resistance of transgenic plants expressing CytoRP to model viruses
containing a TLS is determined. For this, transgenic plants expressing CytoRP as well
as control wild-type plants are infected with preparations of TLS (RNA) viruses. After
infection, a comparative quantitative analysis of viral titer is performed over time.
Viral titer is followed by immuno-detection using antibodies specific for viral proteins.
2. Results
[0074] To determine the localization of CytoRP
in vivo, its cDNA was cloned into the vector pART7eYFP, thus inducing the fusion of CytoRP
with the fluorescent protein eYFP (Gleave, 1992). Protoplasts of
Arabidopsis cells were transformed transiently with the construct expressing the CytoRP-eYFP
fusion. eYFP fluorescence was visualized by confocal laser scanning microscopy using
a Zeiss LSM510 microscope. This revealed that the CytoRP protein is indeed localized
in vivo in the cytosol of
Arabidopsis cells (Figure 3 and Figure 4).
[0075] As a second step, it was built plants with CytoRP stably encoded in the genome. For
this, a DNA fragment was generated where the CytoRP cDNA is inserted between the promoter
sequence of AtPRORP2
in vivo (positions -1000 to -1 upstream of the native of AtPRORP2 initiation codon) and the
terminator sequence of AtPRORP2
in vivo (positions +1 to +118 downstream of the AtPRORP2 terminaton codon). Promoter and
terminator sequences were amplified from
Arabidopsis thaliana genomic DNA. The resulting fragment was cloned in the binary vector pGWB13 (Nakagawa
et al., 2007). The construct obtained was used to transform
Arabidopsis thaliana ecotype col0 plants by the "floral dip" method (Clough and Bent, 1998). Transformed
plants coding for CytoRP were identified by PCR using total genomic DNA from transformed
plants extracts (Figure 5). The selected plants thus contain all the PRORP genes encoded
by the nuclear genome as well as CytoRP, expressed at the same level as AtPRORP2 and
located in the cytosol.
[0076] Despite the removal of the NLS domain from AtPRORP2, CytoRP retains all the elements
necessary for RNase P activity, especially the PPR domain responsible for RNA substrates
binding and the NYN domain responsible for the catalytic activity of PRORP.
[0077] Transgenic
Arabidopsis plants expressing CytoRP, a protein localized in the cytosol and holding RNase P
activity, were constructed. This activity leads to the cleavage of tRNA-like structures
(TLS) of plant viruses and thus generates plant resistance to TLS containing viruses.
[0078] RNase P activity assays of Arabidopsis CytoRP on
in vitro synthesized transcripts representing tRNA like structures of plant viruses were carried
out. Transcripts representing the 3' termini of TYMV, TMV and BMV genomic RNA tRNA
like structures (TLS) were generated by T7 transcription
in vitro and put in presence of Arabidopsis CytoRP proteins to test for RNase activity. The
results are shown in Figure 6.
EXAMPLE II: AMPLIFICATION AND CLONING OF CYTORP CDNA SEQUENCES OF REPRESENTATIVE SPECIES
OF AGRONOMICAL INTEREST.
[0079] The CytoRP sequences from various agronomic relevant plants were amplified using
the primers containing the restriction site
NcoI (CCATGG) and
XhoI (CTCGAG) for digestion and ligation in the plasmid pET28b. These sequences and primers
were as follow:
Tobacco
[0080] Nicotiana tabacum CytoRP based on NtPRORP1 cv Samsun NN, genome "T" (mts deleted): SEQ ID NO: 240.
Primer forward: SEQ ID NO: 241
Primer reverse: SEQ ID NO: 242
[0081] The deleted part of NtPRORP1 (genome T) gene is presented in SEQ ID NO: 243. Only
the 5' (N-terminus) of the gene (protein) is presented in SEQ ID NO: 243. The 3' (C-terminus)
was not changed except the removal of the stop codon to fuse the gene with a 6xHis
tag.
Cucumber
[0082] Cucumis sativus CytoRP based on CsPRORP3 (N-terminus nls deleted): SEQ ID NO: 245.
Primer forward: SEQ ID NO: 246
Primer reverse: SEQ ID NO: 247
[0083] The deleted part of CsPRORP3 gene is presented in SEQ ID NO: 248. Only the 5' (N-terminus)
of the gene (protein) is presented in SEQ ID NO: 248. The start codon was followed
by ggc for glycine in order to accommodate the
NcoI restriction site. The 3' (C-terminus) was not changed except the removal of the
stop codon to fuse the gene with a 6xHis tag.
Tomato
[0084] Solanum lycopersicum CytoRP based on S1PRORP3 (N-terminus nls deleted): SEQ ID NO: 250
Primer forward: SEQ ID NO: 251
Primer reverse: SEQ ID NO: 252
[0085] The deleted part of S1PRORP3 gene is presented in SEQ ID NO: 253. Only the 5' (N-terminus)
of the gene (protein) is presented in SEQ ID NO: 253. The 3' (C-terminus) was not
changed except the removal of the stop codon to fuse the gene with a 6xHis tag.
Lettuce
[0086] Lactica sativa CytoRP based on LsPRORP3 (N- & C-termini nls deleted): SEQ ID NO: 255
Primer forward: SEQ ID NO: 256
Primer reverse: SEQ ID NO: 257
[0087] The deleted parts of the LsPRORP3 gene are presented in SEQ ID NO: 258 (N-terminus)
and SEQ ID NO 260 (C-terminus). Only the 5' (N-terminus) and 3' (C-terminus) of the
gene (protein) are presented SEQ ID NO: 258 and SEQ ID NO 260 respectively. The remaining
part of the gene (protein) was not changed.
Onion
[0088] Allium cepa CytoRP based on AcPRORP3 (C-terminus nls deleted): SEQ ID NO: 262.
Primer forward: SEQ ID NO: 263
Primer reverse: SEQ ID NO: 264
[0089] The deleted part of AcPRORP3 gene is presented in SEQ ID NO: 265. Only the 3' (C-terminus)
of the gene (protein) is presented in SEQ ID NO: 265. The 5' (N-terminus) was not
changed except the addition of a gcg codon for alanine directly after the start codon
of the gene contained in the
NcoI restriction site.
EXAMPLE III: TOTAL RNA EXTRACTION FROM PLANTS, DNASE TREATMENT AND REVERSE TRANSCRIPTION
[0090] TRIzol® RNA Isolation Reagents (LifeTechnology) was used to extract RNA from plant
samples.
[0091] Plants material was leaves from each plant.
[0092] Mortar and pestle were frozen using liquid nitrogen.
[0093] Leaf material (about 3 g) was ground to powder in liquid nitrogen.
[0094] Then, TRIzol (3 to 4 ml) was added to the powder. The powder was mixed with the TRIzol
by inverting the tube and left 5 minutes on the bench at room temperature. Aliquotes
of 1 ml were transferred in 2 ml tubes and 0.2 ml chloroform was added and the tubes
were put on vortex thoroughly for 1 min. Then, the tubes were left 5 minutes on the
bench at room temperature and centrifuged full speed for 10 min at 4°C. The supernatant
(600 µl) was transferred in a new RNase free tube. 300 µl isopropanol was added, the
tube inverted few times and then left 15 minutes on the bench at room temperature.
The tubes were centrifuged full speed for 15 min at 4°C. The supernatant was removed,
the pellet was washed with 1 ml 75% cold ethanol. The supernatant was removed, the
pellet dried and resuspended in 20 µl RNase free mQ water.
[0095] Total RNA concentration was determined using the nanodrop 2000 (Thermo Scientific).
[0096] 15 µg or 10 µg of total RNA was used for DNase I treatment in order to get rid of
residual genomic DNA contamination. 10 µl DNase I buffer + MgC12 10x and 10 µl DNase
I (1 unit / µl) (Thermo Scientific) were added in a total volume of 100 µl.
[0097] The tubes were incubated 30 min at 37°C.
[0098] A RNA phenol/chloroform extraction was then operated. 100 µl phenol/chloroform was
added to the reaction and vortex thoroughly for 20 sec. The tubes were centrifuge
full speed at room temperature for 5 min.
[0099] The aqueous supernatant was transferred into a new RNase free tube and the RNA was
precipitated with ethanol (10 µl 3 M Na Acetate pH5.3 and 250 µl absolute ethanol).
[0100] The tubes were left 1 hour at -20°C and then centrifuged full speed for 30 minutes
at 4°C.
[0101] The supernatant was removed, and 1 ml 75% ethanol was added to wash the pellet.
[0102] The tubes were centrifuged full speed for 5 minutes at 4°C and the supernatant removed.
[0103] The pellet was dried and re-suspended in 10 µl RNase free mQ water.
[0104] 3 to 5 µg of total RNA were used for the first strand cDNA synthesis.
[0105] Maxima Reverse Transcriptase (Thermo Scientific) at 200 U/µl supplied with 5x RT
buffer were used.
[0106] A mix of oligo(dT)
18 and random primer was used for the first strand cDNA synthesis.
[0107] The reactions were performed with the provider specifications.
[0108] Typical first strand cDNA synthesis is as follow:
|
1 reaction |
RNA treated DNase I (5 µg) |
5 µl |
Oligo(dT)18 (100 µM) |
0.5 µl |
Random Primers (0,2µg/µl) |
0.5 µl |
dNTP |
1 µl |
H2O |
7.5 µl |
[0109] The PCR tube containing the mix is incubated at 65°C for 5 min then put on ice for
2 min.
[0110] After a short spin in a bench-top centrifuge, the following mix is added in the tube:
Buffer Maxima RT 5x |
4 µl |
RNase OUT 40 U/µl (Invitrogen) |
0.5 µl |
Maxima RT enzyme |
1 µl |
[0111] The PCR tube containing the mix is centrifuged shortly and incubated 10 min at 25°C,
45 min at 50°C and the enzyme is inactivated at 85°C for 5 min.
[0112] The cDNA is then ready for use in PCR reaction.
[0113] 1 to 2 µl cDNA produced were used to amplify PRORP coding sequences with the primers
listed below:
Primers were designed and ordered at Integrated DNA Technologies (IDT) to amplify
cDNA of PRORP from tobacco
Nicotiana tabacum (Nt), cucumber
Cucumis sativus (Cs), lettuce
Lactuca sativa (Ls), tomato
Solanum lycopersicum (S1) and onion
Allium cepa (Ac).
NtP1F |
gtcattcatatccccagcaatg |
SEQ ID NO: 267 |
NtP1R |
ccctcggagtcgatcaatttat |
SEQ ID NO: 268 |
CsP3F |
ctacagatacttctggaatggattc |
SEQ ID NO: 269 |
CsP3R |
ggactcggccacatagcta |
SEQ ID NO: 270 |
LsP3F |
gcaaggagaacttactcaacaatg |
SEQ ID NO: 271 |
LsP3R |
tgtgacaaaaaacccaagtttcta |
SEQ ID NO: 272 |
S1P3F |
gccattactaccggaaaatg |
SEQ ID NO: 273 |
S1P3R |
gttctggaaaaggtatcaccttc |
SEQ ID NO: 274 |
AcP3F |
ctcagtcgacccagaaaagtatg |
SEQ ID NO: 275 |
AcP3R |
caaaactaacgaccacaaaaatgcta |
SEQ ID NO: 276 |
[0114] Typical PCR mix is as follow:
Components for 1 PCR reaction (µl)
2x Phusion MasterMix (Thermo Fisher Scientific) |
25 |
Forward primer |
2.5 |
Reverse primer |
2.5 |
cDNA |
1 |
mQ H2O |
19 |
Total volume |
50 |
[0115] Typical PCR cycling is as follow:
Initial denturation |
98°C |
30 sec |
Denaturation |
98°C |
10 sec |
Hybridization |
60°C |
10 sec |
Elongation |
72°C |
1 min 30 sec |
Final elongation |
72°C |
5 min |
35 cycles of denaturation, hybridization and elongation were done
[0116] A PCR sample of 2 µl (from the 50 µl) was mixed with water (3 µl) and 6x DNA loading
dye (1 µl) and charged into a 1 % agarose gel. 4.5 µl MassRuler (Thermo Scientific)
was used for size determination. Results are shown in Figure 7.
[0117] The DNA from the remaining of the PCR was extracted (kit Macherey-Nagel referred
as MN hereafter Nucleospin Gel and PCR cleanup).
[0118] The standard protocol of the kit was used and elution was made with 15 µl NE (Tris-HCl
pH8,5).
[0119] The purified DNA was quantified with nanodrop 2000.
[0120] The Phusion polymerase producing blunt ends, a A-tailing procedure was done using
the protocol of "pGEM-T and pGEM-T Easy vector systems" manual (Promega).
[0121] Typical A-tailing procedure is as follow (in 0.2 ml PCR tubes):
Components for 1 reaction (µl):
H2O mQ |
2.8 |
Tampon Taq 10x with MgCl2 |
1 |
dATP (10 mM) |
0.2 |
Cleaned up DNA from PCR |
5 |
GoTaq2 (Promega) |
1 |
Total (µl) |
10 |
[0122] Incubation at 70°C for 20 min in thermocycler.
[0123] The A-tailed product is ligated into the pGEM-T easy vector following the procedure
described in the manual of "pGEM-T and pGEM-T Easy vector systems".
[0124] Typical ligation procedure is as follow (in 0.5 ml tubes):
Component for 1 ligation (µl):
2x rapid ligase buffer |
2.5 |
pGEM-T easy vector |
0.5 |
A-tailed DNA from PCR |
1.5 |
T4 DNA ligase |
0.5 |
Total (µl |
5 |
[0125] The tubes were incubated for 3 hours or overnight at room temperature.
[0126] The ligation mix was used for
E. coli TOP10 chemo-competent cells transformation.
[0127] Typical transformation procedure is as follow (in 0.5 ml tubes):
-80°C conserved E. coli TOP10 chemo-competent cells were thawed on ice for 15 min.
2.5 µl of ligation mix is added to the cells in ice and left for 30 min in ice.
[0128] Heat shock at 42°C was performed for 45 sec (water bath).
[0129] The tubes were then cool down 2 min in ice.
[0130] 600 µl sterile LB solution was added to the cell transferred into a 13 ml round bottom
tube.
[0131] The tubes are incubated at 37°C on a shaker for 1 hour.
[0132] 200 µl cells are plated on Petri dish containing 25 ml LB agar supplemented with
ampicillin and X-gal (in flow hood).
[0133] After drying, the plates are incubated at 37°C for the night.
[0134] The next morning, plates are placed in the fridge to increase the blue-white screening
of the colonies.
[0135] The white colonies (containing an insertion in the
LacZ gene) are used for a PCR screening.
[0136] Typical PCR reaction is as follow:
Components for 1 reaction (µl)
H2O mQ |
13.4 |
Tampon GoTaq 5x with LD (Promega) |
4 |
MgC12 (25 mM) (Promega) |
1.2 |
dNTPs (10 mM) |
0.4 |
M13 FW (10 uM) |
0.4 |
M13 RV (10 uM) |
0.4 |
GoTaq 2 enzyme (Promega) |
0.2 |
Bacteria from a single colony |
bacteria |
Total volume |
20 |
[0137] Master mixes were prepared to screen for 8 to 16 colonies
[0138] Typical PCR cycling is as follow:
Initial denaturation |
95°C |
3 min |
Denaturation |
95°C |
30 sec |
Hybridization |
47°C |
30 sec |
Elongation |
72°C |
2 min 30 sec |
Final elongation |
72°C |
5 min |
35 cycles of denaturation, hybridization and elongation were done
[0139] 1% agarose gel is prepared and 5 µl PCR product was loaded. 4.5 µl Mass-Ruler was
added for size determination. Results are shown in Figure 8.
[0140] Plasmid preparations were performed with 3 ml LB ampicillin cultures inoculated with
colonies containing CytoRP (overnight cultures). Kit MN, Nucleospin Plasmid QuickPure
(Elutions with 30 µl NE).
[0141] The concentration of these samples was determined with Nanodrop 2000.
[0142] Sequence analysis revealed that no single nucleotide polymorphism for the various
sequences that could alter the production of the CytoRP is present. Then, the positive
plasmids were diluted to 5 ng/µl and were used as PCR templates for the production
of the respective CytoRP. For
N. tabacum only a PRORP1 clone was used to produce a CytoRP (not PRORP3).
[0143] The primers presented in Example II were used to amplify the CytoRP genes in order
to clone them in pET28b expression plasmid.
[0144] Typical PCR mix is as follow:
Components for 1 PCR reaction (µl)
2x Phusion MasterMix (Thermo Fisher Scientific) |
25 |
Forward primer |
2.5 |
Reverse primer |
2.5 |
Plasmid (5 ng/µl) |
1 |
mQ H2O |
19 |
Total volume |
50 |
[0145] Typical PCR cycling is as follow:
Initial denturation |
98°C |
30 sec |
Denaturation |
98°C |
10 sec |
Hybridization |
60°C |
10 sec |
Elongation |
72°C |
1 min 30 sec |
Final elongation |
72°C |
5 min |
35 cycles of denaturation, hybridization and elongation were done.
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